2] l-Glutamate decarboxylase from bacteria

Abstract

Bacterial L-glutamate decarboxylase (L-glutamate l-carboxy-lyase) is purified to homogeneity from Escherichia coli (E. coli) and from Clostridium perfringens. When E. coli is grown in the medium containing L-glutamate, the inducible enzyme is produced in large amounts as the medium becomes acidic. The enzyme activity may be determined using a manometric assay of CO2 evolution or a radiometric assay of the conversion of [14C]glutamate to 14CO2 or γ-[14C]aminobutyrate. The manometric assay is used to measure enzyme activity during purification and in steady state kinetic studies. Chloride, an activator, and the coenzyme are added to the assay mixture to produce optimum conditions. All the steps except for the heat step—in the procedure for the growth of the E. coli and isolation of the glutamate decarboxylase—are carried out at 4°. After the second ammonium sulfate precipitation, the enzyme is protected from light. Typical results of purification of the enzyme from E. coli harvested from 90 liters of medium or approximately 500 g of cell paste are presented in a tabulated form in this chapter.